Project Summary/Abstract The goal of this proposal is to investigate the intrinsic ability of hematopoietic transcription factors (TFs) to engage closed chromatin and initiate chromatin opening during cell fate changes. Cell fate control is a fundamental process in biology and de novo generation of clinically relevant cell types has enormous implications for research and therapeutics; however, the general principles by which closed and unmarked chromatin is initially accessed by these factors are not fully understood. In eukaryotic cells, chromatin compaction serves as a mechanism for gene regulation by modulating the accessibility of TFs to target DNA sequences. Development of macrophages from hematopoietic stem cells requires coordinated expression of PU.1 with myeloid TFs C/EBP? and C/EBP?, and ectopic expression of PU.1 and C/EBP?/? converts fibroblasts to the macrophage lineage. I hypothesized that PU.1, C/EBP?, and C/EBP? act as ?pioneer? factors and that initiating development requires these factors to directly bind compacted chromatin, initiate chromatin opening, and allow subsequent binding of additional TFs that activate gene expression. Our lab identified nucleosomes targeted by PU.1, C/EBP?, and C/EBP? in vivo, and reconstituted these nucleosomes in vitro. To assess the interactions of these factors with higher-order chromatin substrates, I reconstituted fluorescently end-labeled synthetic nucleosome arrays and inserted a central nucleosome capable of binding by PU.1, C/EBP?, and C/EBP?. I have found that PU.1, C/EBP?, and C/EBP? bind and initiate accessibility of H1- compacted nucleosome arrays with different efficiencies. Furthermore, I have found that the DNA binding domains of PU.1 and C/EBP? open chromatin with less efficiency compared to the full-length proteins. I am now investigating the ability of these pioneer factors to open chromatin and how their mechanisms of action compare to another established pioneer factor, FoxA. I hypothesize that PU.1, C/EBP?, and C/EBP? possess protein domains that enable chromatin interactions, and that initiating chromatin accessibility is fundamental to the ability of these factors to direct cell fate in vivo. To address this hypothesis I will 1) use nanopore sequencing to define TF-chromatin interactions and determine if PU.1, C/EBP?, and C/EBP? interact with histones using crosslinking mass spectrometry 2) perform a deletion analysis of PU.1, C/EBP?, and C/EBP? to map domains required for chromatin opening in vitro and determine if chromatin opening by pioneer factors is required for TF directed macrophage transdifferentiaiton. My project will provide insights into the mechanisms of chromatin structure regulation by hematopoietic TFs during cellular development and reprogramming. Furthermore, biochemical analysis will provide details of the molecular features that endow TFs with pioneering activity.